Project Summary Chronically high levels of the stress hormone cortisol are deleterious to many organs systems. ACTH- independent Cushing's syndrome is an endocrine disorder wherein the adrenal glands constitutively produce excess cortisol. Symptoms include obesity, thinning of the skin, cognitive and emotional problems, and bone loss. Recent studies have identified a mutation in the protein kinase A catalytic subunit (PKAc) in approximately 50% of these cases. This mutation, L205R, is on the face of PKAc that binds to its regulatory subunit, and is predicted to disrupt holoenzyme formation. Traditionally, the model for cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling has relied on evidence using supraphysiological levels of cAMP stimulation, which leads to full dissociation of PKAc from its regulatory subunit and from A-kinase anchoring proteins (AKAPs). Recent evidence shows that physiological levels of cAMP do not dissociate PKA holoenzymes, thereby redefining the model of PKA activation in cells. Based on this new information, the proposed experiments will test if Cushing's syndrome mutations in PKAc disrupt localization of active PKA and cause ectopic phosphorylation of substrates and downstream cortisol secretion. The hypothesis will be tested in two aims: Aim 1: Is the spatiotemporal profile of PKAc L205R altered in Cushing's syndrome? A combined strategy of CRISPR/Cas9 gene-editing and live-cell imaging with photoactivatable fluorophores and FRET-based biosensors will ascertain: 1) if mutant PKAc is recruited to AKAP-signaling islands, 2) whether these Cushing's mutants are more mobile inside cells, and 3) if PKAc activity aberrantly accumulates at subcellular regions. Aim 2: How does mutant PKAc cause excess cortisol production? I will use chemical biology techniques combined with cortisol measurement in NCI-H295R adrenal cells 1) to determine if mislocalization of PKAc activity is necessary and sufficient to boost cortisol release. Next, by combining miniTurbo proximity labeling with phospho-proteomics, I will establish 2) if the disease-causing L205R PKAc mutant displays altered substrate selectivity that adversely impacts downstream signaling. Additionally, a mouse model of adrenal PKAc L205R expression will be generated to evaluate candidate therapeutic intervention strategies resulting from this aim. The long-term goals of this research plan are to elucidate molecular mechanisms of the signaling underlying hypercortisolism in disease and to spur development of new therapeutic tools. The research will be conducted at the University of Washington in the Department of Pharmacology. This environment provides excellent training for academic-track postdoctoral researchers. Training benefits include strong collaboration within and among departments, approachable principal investigators performing innovative work, and frequent seminars from multiple departments featuring world experts in their respective fields.